Sonic hedgehog (Shh) is a signaling molecule that has multiple critical functions during development, including in organogenesis of the lungs, pancreas, and intestine. Shh is expressed in the pharyngeal endoderm, but its role in the embryonic development of pharyngeal organs has not been investigated. We are particularly interested in the molecular mechanisms controlling organogenesis of the thymus and parathyroids, which originate in the 3rd pharyngeal pouch. Although we are beginning to identify the transcription factors that control imitation and early development of these organs, little is known about the signaling molecules that mediate these early events, or about the pathways controlling later differentiation of these organs. Our preliminary analysis of the Shh mutant mouse suggests that Shh has differential roles in initial thymus/parathyroid organogenesis and fetal thymic epithelial cell (TEC) differentiation. The proposed experiments will test the hypothesis that dynamic regulation of Shh expression is required for initiation of thymus organogenesis, regionalization of the thymus/parathyroid primordium, and normal TEC differentiation. Our specific aims are to: 1) analyze the thymus/parathyroid organogenesis phenotype of the Shh-l- mouse using histological and gene expression studies; and 2) develop a novel embryo culture system and new transgenic mouse strains to test the role of Shh during initial organogenesis and fetal TEC differentiation. The proposed experiments will serve as a pilot study for our long-range goal of identifying and studying the signaling pathways that control pharyngeal region development, and will generate critical new reagents and methodologies that will form the basis for future studies. Analysis of the Shh mutant phenotype proposed in Aim 1) it is necessary to formulate a working model of how Shh is acting at different stages of pharyngeal development and organogenesis. The development of new transgenic mouse strains and embryo culture techniques in Aim 2 will provide essential tools for in-depth analysis of the function of Shh in the thymus. These tools will also allow us to expand our investigation to other signaling pathways, such as BMPs and FGFs, that are known to interact with the Shh pathway in other organs.